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:root {
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li p.first {
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ol {
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ol:first-child {
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table tr td {
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<div  id='write'  class = 'is-mac'><h1><a name="automating-gene-family-evolution-analyses" class="md-header-anchor"></a><span>Automating gene family evolution analyses</span></h1><p><strong><em><span>This tutorial takes a list of homologous genes of interest, BLASTs to obtain these sequences from NCBI, combines the outputs, and aligns them with ClustalW.</span></em></strong><span> </span><em><span>Here we use this protocol to get 10 Toll-Like Receptor genes from tetrapods with available genomes on NCBI (N=22). We divide the sequences into TLR gene subfamilies (known a priori), and automatically aligin the subfamily sequences into alignments  to use for phylogenetic reconstruction</span></em></p><p>&nbsp;</p><h3><a name="step-1:-clone-github-repo" class="md-header-anchor"></a><span>Step 1: Clone GitHub repo</span></h3><p><em><span>The scripts will run straight out of the box (note—these were only tested on unix and linux platforms)</span></em></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; <span class="cm-builtin">git</span> clone https://github.com/mmoral31/GopAga2.0-Genome.git</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p>&nbsp;</p><h3><a name="step-2:-make-a-blast-database-for-your-species-of-interest-(i.e.-target-species)" class="md-header-anchor"></a><span>Step 2: Make a BLAST database for your species of interest (i.e. target species)</span></h3><blockquote><p><span>Requires BLAST+</span></p></blockquote><p><span>if you want to query against whole proteomes, search for the target species on NCBI and pull protein FASTAs (e.g., </span><em><span>Chelonia mydas</span></em><span>)</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; makeblastdb <span class="cm-attribute">-in</span> GCF_000344595.1_CheMyd_1.0_protein.faa <span class="cm-attribute">-dbtype</span> prot <span class="cm-attribute">-parse_seqids</span> <span class="cm-attribute">-out</span> CmydPro</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 44px;"></div><div class="CodeMirror-gutters" style="display: none; height: 44px;"></div></div></div></pre><p><strong><em><span>[repeat for each species of interest]</span></em></strong></p><p>&nbsp;</p><h3><a name="step-3:-set-up-query-sequences-and-input-file" class="md-header-anchor"></a><span>Step 3: Set up query sequences and input file</span></h3><p><strong><span>Need to:</span></strong><span> </span></p><p><strong><span>1) combine query sequences into a single FASTA</span></strong></p><p><span>	</span><em><span>in this case we’re using TLR protein sequences from human. You want to pick a query taxon that is well curated (i.e. will have the most complete suite of genes with the best orthology assignments)</span></em></p><p><strong><span>2) set up input text file with gene names</span></strong></p><p><span>determine the query sequences to be used and place into a single FASTA file labeled as {gene family}_query.fasta where {gene family} is the name given to the group of genes used as a query (e.g., TLR_query.fasta)</span></p><p><span>e.g., </span><strong><em><span>query genes:</span></em></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="" style="page-break-inside: unset;"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang=""><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation" style=""><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt;NP_003256.1 toll-like receptor 3 precursor [Homo sapiens]</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">MRQTLPCIYFWGGLLPFGMLCASSTTKCTVSHEVADCSHLKLTQVPDDLPTNITVLNLTHNQLRRLPAAN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">FTRYSQLTSLDVGFNTISKLEPELCQKLPMLKVLNLQHNELSQLSDKTFAFCTNLTELHLMSNSIQKIKN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">NPFVKQKNLITLDLSHNGLSSTKLGTQVQLENLQELLLSNNKIQALKSEELDIFANSSLKKLELSSNQIK</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">EFSPGCFHAIGRLFGLFLNNVQLGPSLTEKLCLELANTSIRNLSLSNSQLSTTSNTTFLGLKWTNLTMLD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LSYNNLNVVGNDSFAWLPQLEYFFLEYNNIQHLFSHSLHGLFNVRYLNLKRSFTKQSISLASLPKIDDFS</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">FQWLKCLEHLNMEDNDIPGIKSNMFTGLINLKYLSLSNSFTSLRTLTNETFVSLAHSPLHILNLTKNKIS</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">KIESDAFSWLGHLEVLDLGLNEIGQELTGQEWRGLENIFEIYLSYNKYLQLTRNSFALVPSLQRLMLRRV</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ALKNVDSSPSPFQPLRNLTILDLSNNNIANINDDMLEGLEKLEILDLQHNNLARLWKHANPGGPIYFLKG</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LSHLHILNLESNGFDEIPVEVFKDLFELKIIDLGLNNLNTLPASVFNNQVSLKSLNLQKNLITSVEKKVF</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">GPAFRNLTELDMRFNPFDCTCESIAWFVNWINETHTNIPELSSHYLCNTPPHYHGFPVRLFDTSSCKDSA</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">PFELFFMINTSILLIFIFIVLLIHFEGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWV</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">WEHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAI</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">EQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt;NP_003257.1 toll-like receptor 4 isoform C [Homo sapiens]</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">MELNFYKIPDNLPFSTKNLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILT</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">GNPIQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLTNLEHL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">DLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLRNNFDSLNVMKTCIQGL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">AGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSSFSLVSVTI</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ERVKDFSYNFGWQHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGC</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">CSQSDFGTTSLKYLDLSFNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTR</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">VAFNGIFNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVLNMSHN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">NFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFACTCEHQSFLQWIKDQRQL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRG</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQH</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">FIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRR</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LRKALLDGKSWNPEGTVGTGCNWQEATSI</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt;NP_057646.1 toll-like receptor 7 precursor [Homo sapiens]</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIPGGIPTNTTNL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKPRSFSGLTYLKSLYLDGNQLL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">EIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLGQNCYYRNPCYVSYSIEKDAFLNLTKLKVLS</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LKDNNVTAVPTVLPSTLTELYLYNNMIAKIQEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQ</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">IPVNAFDALTELKVLRLHSNSLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">FELQVYRASMNLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNKEASFMSVNES</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">CYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQPLAELRYLDFSNNRLDLLHST</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">AFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQKLMMNDNDISSSTSRTMESESLRTLEFRGNH</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LDVLWREGDNRYLQLFKNLLKLEELDISKNSLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">LETLDLSHNQLTTVPERLSNCSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPEN</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">VLNNLKMLLLHHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTKDPAVTEWVLA</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">ELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDKYAKTENFKIAFYLSHQRLMD</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">EKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQAHPYFWQCLKNALATDNHVAYSQVFKETV</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 946px;"></div><div class="CodeMirror-gutters" style="display: none; height: 946px;"></div></div></div></pre><p><span>create a text file with each line containing the name of one gene family member in the order of the reference FASTA (e.g., TLR.txt)</span></p><p><span>e.g., </span><strong><em><span>gene family text file:</span></em></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang=""><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang=""><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR3</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR4</span></pre><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR7</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 66px;"></div><div class="CodeMirror-gutters" style="display: none; height: 66px;"></div></div></div></pre><p>&nbsp;</p><h3><a name="step-4:-use-_blastp.py_-to-extract-query-genes-from-the-target-species" class="md-header-anchor"></a><span>Step 4: Use </span><em><span>blastp.py</span></em><span> to extract query genes from the target species</span></h3><blockquote><p><span>Requires BLAST+ and Biopython</span></p></blockquote><p><em><span>blastp.py</span></em><span> takes three parameters in this order:</span></p><ul><li><span>name of the created BLAST database </span><em><span>without suffix</span></em><span> (</span><strong><span>e.g., CmydPro</span></strong><span>)</span></li><li><span>name of the gene family for query (e.g., tlr). This is important for distinguishing the reference FASTA (</span><strong><span>e.g., TLR_query.fasta</span></strong><span>)</span></li><li><span>name of the gene family text file (</span><strong><span>e.g., TLR.txt</span></strong><span>)</span></li></ul><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; python blastp.py CmydPro tlr TLR.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>this will output one file per gene family member to record standard output BLAST results and one aggregate file with all pulled sequences from the target species.</span></p><ul><li><span>{gene family member}_{db name} for all of the individual records</span></li><li><span>{gene family}_{db name}.fa for the aggregate FASTA file (</span><strong><span>e.g., TLR_CmydPro.fa</span></strong><span>)</span></li></ul><p>&nbsp;</p><h3><a name="step-5:-use-_pull_id_match.py_-to-separate-output-into-subfamilies-for-analysis" class="md-header-anchor"></a><span>Step 5: Use </span><em><span>pull_id_match.py</span></em><span> to separate output into subfamilies for analysis</span></h3><blockquote><p><span>Requires Biopython</span></p></blockquote><p><span>if the gene family of interest has subfamilies you want to align and analyze separately (as in TLRs), use pull_seqids.py to separate and extract a subfamily of interest </span><em><span>[if you don’t want to subset your data you can skip this step]</span></em></p><p><em><span>pull_id_match.py</span></em><span> takes three parameters in this order:</span></p><ul><li><span>name of the FASTA file to extract from (</span><strong><span>e.g., TLR_CmydPro.fa</span></strong><span>)</span></li><li><span>name of the text file with listed keywords to pull with (</span><strong><span>e.g., TLR3SF.txt</span></strong><span>)</span></li><li><span>name of the output file where pulled sequences will aggregate (</span><strong><span>e.g., TLR3SF.fa</span></strong><span>)</span></li></ul><p><span>The text file will have one keyword on each line. The FASTA output will include any records that matched at least one of the keywords in the list.</span></p><p><span>e.g., </span><strong><span>text file:</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang=""><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang=""><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR3</span></pre></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR4</span></pre></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 44px;"></div><div class="CodeMirror-gutters" style="display: none; height: 44px;"></div></div></div></pre><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; python pull_id_match.py TLR_CmydPro.fa TLR3SF.txt TLR3SF.fa</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p>&nbsp;</p><h3><a name="step-6:-use-_run_alignments.py_-to-align-respective-subfamilies-with-clustalw" class="md-header-anchor"></a><span>Step 6: Use </span><em><span>run_alignments.py</span></em><span> to align respective subfamilies with ClustalW</span></h3><blockquote><p><span>Requires ClustalW2</span></p></blockquote><p><span>to align subfamilies for further analysis (e.g., tree reconstructions) use </span><strong><em><span>run_alignments.py</span></em></strong><span> </span></p><p><em><span>run_alignments.py</span></em><span> takes one parameter:</span></p><ul><li><span>name of the text file with listed names of FASTA files (without suffix) to be aligned (e.g., TLR_align.txt) </span><em><span>Note—these files will be aligned separately. This is useful if you want to make many separate alignments</span></em></li></ul><p><span>e.g., </span><strong><span>text file:</span></strong></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang=""><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang=""><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">TLR3SF</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; python run_alignments.py TLR_align.txt</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p><span>three alignment files are output in three formats: FASTA, NEXUS, PHYLIP</span></p><p><em><span>Note—ClustalW needs to be in your Bash profile path to be recognized</span></em></p><p>&nbsp;</p><h3><a name="step-7:-determine-substitution-model" class="md-header-anchor"></a><span>Step 7: Determine substitution model</span></h3><p><span>Here we used Prottest to determine the best substitution matrix for tree reconstruction</span></p><blockquote><p><span>Requires ProtTest</span></p></blockquote><p><span>Prottest can be used to determine the best substitution matrix for your data. Requires PHYLIP alignment as input.</span></p><pre spellcheck="false" class="md-fences md-end-block ty-contain-cm modeLoaded" lang="bash"><div class="CodeMirror cm-s-inner CodeMirror-wrap" lang="bash"><div style="overflow: hidden; position: relative; width: 3px; height: 0px; top: 0px; left: 7.5px;"><textarea autocorrect="off" autocapitalize="off" spellcheck="false" tabindex="0" style="position: absolute; bottom: -1em; padding: 0px; width: 1000px; height: 1em; outline: none;"></textarea></div><div class="CodeMirror-scrollbar-filler" cm-not-content="true"></div><div class="CodeMirror-gutter-filler" cm-not-content="true"></div><div class="CodeMirror-scroll" tabindex="-1"><div class="CodeMirror-sizer" style="margin-left: 0px; margin-bottom: 0px; border-right-width: 0px; padding-right: 0px; padding-bottom: 0px;"><div style="position: relative; top: 0px;"><div class="CodeMirror-lines" role="presentation"><div role="presentation" style="position: relative; outline: none;"><div class="CodeMirror-measure"><pre><span>xxxxxxxxxx</span></pre></div><div class="CodeMirror-measure"></div><div style="position: relative; z-index: 1;"></div><div class="CodeMirror-code" role="presentation"><div class="CodeMirror-activeline" style="position: relative;"><div class="CodeMirror-activeline-background CodeMirror-linebackground"></div><div class="CodeMirror-gutter-background CodeMirror-activeline-gutter" style="left: 0px; width: 0px;"></div><pre class=" CodeMirror-line " role="presentation"><span role="presentation" style="padding-right: 0.1px;">&gt; prottest3 <span class="cm-attribute">-i</span> TLR3SF_alignment.phy <span class="cm-attribute">-o</span> TLR3SF_models</span></pre></div></div></div></div></div></div><div style="position: absolute; height: 0px; width: 1px; border-bottom-width: 0px; border-bottom-style: solid; border-bottom-color: transparent; top: 22px;"></div><div class="CodeMirror-gutters" style="display: none; height: 22px;"></div></div></div></pre><p>&nbsp;</p><h3><a name="step-8:-generate-gene-family-trees" class="md-header-anchor"></a><span>Step 8: Generate gene family trees </span></h3><p><span>Here, we used the CIPRES Science Gateway </span><a href='https://www.phylo.org/portal2' target='_blank' class='url'>https://www.phylo.org/portal2</a><span> (running RaxxML and MrBayes) to generate ML and Bayesian subfamily trees.</span></p><p>&nbsp;</p></div>
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